Korean J Physiol Pharmacol.  2020 Jul;24(4):363-372. 10.4196/kjpp.2020.24.4.363.

Gardenia jasminoides extract and its constituent, genipin, inhibit activation of CD3/CD28 co-stimulated CD4+ T cells via ORAI1 channel

• Kim HJ
• Nam YR
• Woo J
• Kim WK
• Nam JH

• KMID: 2503329
• DOI: http://doi.org/10.4196/kjpp.2020.24.4.363

Abstract

Gardenia jasminoides (GJ) is a widely used herbal medicine with antiinflammatory properties, but its effects on the ORAI1 channel, which is important in generating intracellular calcium signaling for T cell activation, remain unknown. In this study, we investigated whether 70% ethanolic GJ extract (GJ_EtOH) and its subsequent fractions inhibit ORAI1 and determined which constituents contributed to this effect. Whole-cell patch clamp analysis revealed that GJ_EtOH (64.7% ± 3.83% inhibition at 0.1 mg/ml) and all its fractions showed inhibitory effects on the ORAI1 channel. Among the GJ fractions, the hexane fraction (GJ_HEX, 66.8% ± 9.95% at 0.1 mg/ml) had the most potent inhibitory effects in hORAI1-hSTIM1 co-transfected HEK293T cells. Chemical constituent analysis revealed that the strong ORAI1 inhibitory effect of GJ_HEX was due to linoleic acid, and in other fractions, we found that genipin inhibited ORAI1. Genipin significantly inhibited I_ORAI1 and interleukin-2 production in CD3/ CD28-stimulated Jurkat T lymphocytes by 35.9% ± 3.02% and 54.7% ± 1.32% at 30 μM, respectively. Furthermore, the same genipin concentration inhibited the proliferation of human primary CD4⁺ T lymphocytes stimulated with CD3/CD28 antibodies by 54.9% ± 8.22%, as evaluated by carboxyfluorescein succinimidyl ester assay. Our findings suggest that genipin may be one of the active components of GJ responsible for T cell suppression, which is partially mediated by activation of the ORAI1 channel. This study helps us understand the mechanisms of GJ in the treatment of inflammatory diseases.

Keyword

CD4 positive T lymphocytes; Gardenia; Genipin; Interleukin-2; ORAI1 protein

Figure

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  Fig. 1 Inhibitory effects of 70% ethanolic extract of Gardenia jasminoides (GJEtOH) on ORAI1 current (IORAI1) in human STIM1 and ORAI1 co-transfected HEK293T cells. (A) Representative chart trace recordings of IORAI1 and inhibition of IORAI1 activity by 0.1 and 0.3 mg/ml GJEtOH and 10 μM BTP2. (B) Related current (I)-voltage (V) relationship graph at steady-state IORAI1 (1), 0.1 (2), 0.3 (3) mg/ml GJEtOH and 10 μM BTP2 (4). (C) Graph shows inhibition of IORAI1 using 0.03, 0.1, and 0.3 mg/ml GJEtOH and 10 μM BTP2, compared with normalized currents at –120 mV (n = 11). ****p < 0.0001 vs. control.

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  Fig. 2 Seventy percent ethanolic extract of Gardenia jasminoides (GJEtOH) solvent fractions inhibit ORAI1 current (IORAI1). (A) Normalized columnar analysis of IORAI1 inhibition using 0.1 mg/ml water (H2O), butanol (BuOH), ethyl acetate (EtOAc), and hexane (HEX) fractions at –120 mV (n = 4). **p < 0.01 and ****p < 0.0001 vs. the control. (B) Different concentrations of hexane fraction result in varying rates of IORAI1 inhibition at −120 mV (n = 6). ***p < 0.001 and ****p < 0.0001 vs. control.

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  Fig. 3 Chromatograms of hexane fractions of 70% ethanolic extract of Gardenia jasminoides (GJ) generated using gas chromatography mass spectrometry (GC/MS). The peak number indicates putative identification of known compounds with matching degree of over 95%. Detailed list of chemical components of GJ is provided in Table 1.

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  Fig. 4 Effects of linoleic acid, palmitic acid, and stigmasterol on ORAI1 current (IORAI1). (A) Representative current (I)-voltage (V) relationship curve showing the potent inhibition of IORAI1 by 10 μM linoleic acid (n = 3). (B) Representative I-V relationship curve of IORAI1 inhibition by 100 μM palmitic acid (n = 3). (C) Representative I-V relationship curve of IORAI1 inhibition by 100 μM stigmasterol (n = 3). (D) Columnar statistical analysis summarizing the results of the effects of palmitic acid (PA) and stigmasterol (St) on IORAI1.

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  Fig. 5 Identification of chemical constituents of 70% ethanolic extract of Gardenia jasminoides (GJEtOH) using high performance liquid chromatography (HPLC). (A, B) HPLC chromatograms of standard chemicals (A), including geniposidic acid, chlorogenic acid, geniposide, genipin, and rutin, and HPLC chromatograms of GJEtOH (B). The detection of chemical constituents in GJEtOH was performed by comparing the retention times of the UV spectral peak with those of the standard chemicals at 238 nm. (C, D) HPLC chromatograms of standard chemicals (C), including crocin and HPLC chromatograms of GJEtOH (D) detected at 440 nm. (E, F) HPLC chromatograms of standard chemicals (E), including crocin II and HPLC chromatograms of GJEtOH (F) detected at 440 nm.

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  Fig. 6 Concentration-dependent inhibitory effects of genipin on ORAI1 current (IORAI1) in STIM1 and ORAI1 co-transfected HEK293T cells. (A) Normalized columnar analysis of the effects of the nine chemical constituents of 70% ethanolic extract of Gardenia jasminoides (GJEtOH) on IORAI1 (n = 6). **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. the control. DMSO, dimethyl sulfoxide; ChlA, chlorogenic acid; GnP, genipin; GeS, geniposide; GeA, geniposidic acid; Ru, rutin; Cr, crocin; Cr2, crocin II; CrA, croceic acid. (B) Normalized histograms of IORAI1 inhibition by 10, 30, 100, and 300 μM genipin (n = 12). ****p < 0.0001 vs. the control.

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  Fig. 7 Inhibition of interleukin-2 (IL-2) secretion by genipin in CD3/CD28 co-stimulated human T lymphocytes. (A) Human naïve CD4+ T lymphocytes were treated with different concentrations of genipin for 72 h; same volume of solvent (dimethyl sulfoxide, DMSO) under the same conditions was used as vehicle control. Cell viability was assessed with a CCK-8 assay. Cell viability of genipin-treated groups was assessed relative to that of control cells normalized as 1 (n = 3). (B) IL-2 levels were measured in culture supernatants of CD3/CD28 co-stimulated Jurkat T cells at 72 h post-treatment with 10 and 30 μM genipin; treatment with 10 μM BTP2 was used as a positive control. Significance of IL-2 secretion inhibition by genipin was compared between the genipin plus CD3/CD28 co-stimulation treatment group and the CD3/CD28 co-stimulation only group (n = 3). **p < 0.01, ****p < 0.0001 vs. the control.

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  Fig. 8 Genipin inhibits the proliferation of CD3/CD28 co-stimulated primary human CD4+ T lymphocytes. (A) A representative plot shows division of human naïve CD4+ T cells as assessed using carboxyfluorescein succinimidyl ester (CFSE) dilution. Human naïve CD4+ T cells were stained with 1 μM CFSE, treated with 10, 30, and 100 μM genipin (GnP), and cultured in anti-CD3 hAb-coated plates with 2 μg/ml anti-CD28. Cell proliferation was assessed by flow cytometry after 72 h of CD3/CD28 receptor stimulation. Human naïve CD4+ T cells stimulated with anti-CD3 and anti-CD28 only were used as negative controls; those treated with 10 μM BTP2 and stimulated with anti-CD3 and anti-CD28, served as positive control. (B) Statistical analysis summarizing the results of three independent experiments and indicating the relative proportion of CD4+ T cells to the whole T cell population induced by CD3/CD28 co-stimulation. *p < 0.05, ****p < 0.0001 vs. the control.

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